HBV (Hepatitis B Virus) is responsible for chronic or acute human hepatitis that may get worse to liver cirrhosis or cancer. It is estimated that about three hundred million people are suffering from hepatitis in the world (Tiollais and Buendia, Sci. Am. 264:48, 1991).
There are three kinds of HBV surface proteins containing different sets of surface antigens. Particularly, these surface antigen proteins includes the Major Protein containing S antigen, the Middle Protein containing S and pre-S2 antigens, and the Large Protein containing S, pre-S2 and pre-S1 antigens (Neurath and Kent, Adv. Virus Res., 34:65–142, 1988). All the surface antigen proteins can induce antibodies that neutralize HBV, and especially, antibodies against HBV pre-S antigen are associated with the elimination of the virus and the recovery from HBV infection, overcoming non-responsiveness to the S antigen (Iwarson et al., J. Med. Virol., 16:89–96, 1985; Itoh et al., Proc. Natl. Acad. Sci. USA, 85:9174–9178, 1986; Budkowska et al., J. Med. Virol., 20:111–125, 1986; Milich et al., Proc. Natl. Acad. Sci. USA, 82:8168–8172, 1985; Milich et al., J. Immunol., 137:315–322, 1986). Unlike pre-S2 or S antigen, pre-S1 antigen is exclusively present in infectious virus particles (Heerman et al., J. Virol., 52:396–402, 1984) and involved in the infection into human liver cells. Thus, it has been reported that monoclonal antibody specific for pre-S1 antigen may efficiently neutralize HBV (Neurath et al., Cell, 46:429, 1986; Pontisso et al., Virology, 173:533, 1989; Neurath et al., Vaccine, 7:234, 1989), and the monoclonal antibody is considered to be useful in the prevention of HBV infection and the treatment of chronic hepatitis B.
So far hepatitis B immunoglobulin has been employed as a preventive for HBV infection, which may protect, for example, a newborn baby from a HBV-positive mother, medical personnel exposed to HBV, and liver transplant patient with chronic HBV-related liver disease (Beasley et al., Lancet, 2:1099, 1983; Todo et al., Hepatology, 13:619, 1991). However, hepatitis B immunoglobulin has some shortcomings such as its limited availability, low specific activity and its possible contamination with infectious agents. Furthermore, it is another disadvantage of hepatitis B immunoglobulin that blood plasma should be continuously supplied.
As an alternative for the hepatitis B immunoglobulin, mouse monoclonal antibodies against HBV surface antigens have been developed. Although the mouse monoclonal antibodies show high affinity for the antigen and can be prepared on a large scale, they induce human anti-mouse antibody response in patients (Shawler et al., J. Immunol., 135:1530, 1985). There were attempts to prepare human monoclonal antibodies, but few of these antibodies showed a high level of affinity.
Instead, humanized antibodies have been developed. Humanized antibody has a high level of affinity and specificity similar to mouse antibodies, whereas its immunogenicity is minimized. Humanized antibody is a hybrid antibody in which CDRs (Complementarity Determining Regions) of a mouse antibody is grafted to a human antibody by genetic engineering technique. It can be easily prepared on a large scale, and hardly elicits immune responses in humans since most of the DNA sequences encoding the humanized antibodies are derived from a human DNA sequence (Riechman et al., Nature, 332:323, 1988; Nakatani et al., Protein Engineering, 7:435, 1994).
To overcome the aforementioned and other disadvantages of mouse or human HBV immunoglobulin, we, the inventors of the present invention, have attempted to prepare humanized antibodies which can be used to prevent HBV infection and to treat chronic hepatitis B. Prior to this invention, we prepared a mouse monoclonal antibody KR127 against HBV surface antigen pre-S1. Additionally, we isolated the genes encoding the heavy and light chain variable regions of KR127 antibody and determined the sequences of the genes (Korea Patent Application No. 1997-30696). The present invention is performed by selecting human immunoglobulin genes homologous to the sequences of KR127 antibody light chain and heavy chain variable regions; constructing the humanized antibody genes; inserting the genes into expression vectors; introducing the vectors into host cells; obtaining humanized antibodies from the culture of the transformed cells; and confirming that the humanized antibodies have high affinity to HBV pre-S1 antigen, similar to the mouse monoclonal antibody KR127.